CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish

Since its first application for site-directed mutagenesis, the CRISPR-Cas9 system has revolutionized genome engineering. Here, we present a validated workflow for the generation of targeted genomic deletions in zebrafish, including the design, cloning, and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotype screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely used approach in functional genetics. For complete details on the use and execution of this protocol, please refer to Amorim et al. (2020).This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. ERC_2015_StG _ 680156 _ZPR). J.B. acknowledges Fundação para a Ciência e a Tecnologia (FCT) for an FCT Scientific Stimulus Grant ( CEECIND/03482/2018 ). J.A. is a PhD fellow from FCT ( SFRH/BD/145110/2019 ). R.B.C. was funded by FCT ( ON2201403-CTO-BPD ) and EMBO (Short-Term Fellowship). We thank Joaquin Letelier, Ana Novoa, and Moises Mallo for important advice establishing the current protocol. We also thank Joana Marques for constant assistance in establishing the protocol